Siderophore-Mediated Iron Acquisition Mechanisms in Vibrio ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1996, p. 928–935 0099-2240/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 62, No. 3

Siderophore-Mediated Iron Acquisition Mechanisms in Vibrio vulnificus Biotype 2 ´ N FOUZ, ELENA ALCAIDE, ELENA G. BIOSCA, BELE

AND

CARMEN AMARO*

Departamento de Microbiologıá y Ecologıá, Universidad de Valencia, Burjassot, Spain Received 7 April 1995/Accepted 29 December 1995

Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans. In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis. The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels. This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation. No alterations in lipopolysaccharide patterns were detected in response to iron stress. Finally, our data suggest that V. vulnificus biotype 2 uses the hydroxamate-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein. In eels, free-iron levels are well below the limits for bacteria to grow (40). Consequently, to survive and grow in this host, a specific mechanism for iron uptake is necessary. Biotype 1 strains produce phenolic and hydroxamic siderophores and inducible outer membrane proteins that constitute an iron uptake mechanism whose relationship to virulence for humans has not been clearly demonstrated (36, 46). The phenolate (named vulnibactin) has recently been characterized as a polyamine-containing siderophore (24). Only virulent strains of this biotype are able to use iron-bound transferrin (22, 37, 38), and whether capsule plays a significant role in iron acquisition from human transferrin is the object of controversy (37, 45). In this study we have investigated the ability of V. vulnificus biotype 2 to obtain iron through different mechanisms and have also evaluated the importance of free iron in vibriosis. With this aim in mind we have centered our investigation on (i) the relationship between iron availability in host fluids and virulence degree in iron-overloaded eels, (ii) the production of siderophores, detected by chemical and biological assays, and their role in iron acquisition from transferrin, and (iii) the alterations in bacterial envelopes in response to iron-restricted conditions. The role of capsule in iron acquisition was also investigated by using capsulated and acapsulated original isolates. Biotype 1 strains from clinical and environmental sources were assayed for comparative purposes.

The species Vibrio vulnificus comprises two biotypes that can be serologically and biochemically differentiated (4, 10, 12, 13, 42). Biotype 1 is an opportunistic human pathogen, avirulent for eels, which can be isolated from estuarine waters and marine animals (25, 26, 41). This biotype shows a dependence between iron availability in body fluids and degree of virulence for humans (44). Biotype 2 is a primary eel pathogen which, up to now, has never been recovered from water samples (12, 23, 42). Some strains of this biotype are highly virulent; doses as low as 10 cells per fish produce a terminal hemorrhagic septicemia in less than 24 h (15). Therefore, the organism grows rapidly and spreads to the major body organs, from which it can be recovered as pure culture when eels are moribund. This biotype is also virulent for mice, and in this host it shows an iron responsiveness similar to that of the classical human pathogen V. vulnificus biotype 1 (7). Thus, strains virulent for eels are virulent for mice, and hemin, hemoglobin, and Desferal (deferrioxamine B mesylate, a siderophore produced by Streptomyces spp.) enhance their growth rate in fresh human serum and reduce their 50% lethal doses (LD50) in iron-overloaded mice (7). In fact, the possible implication of strains of biotype 2 in one human infection developed after manipulation of eels has been suggested very recently (19). Little is known about the virulence mechanisms of V. vulnificus biotype 2 responsible for vibriosis in eels. The organism shifts between encapsulated and unencapsulated forms (15), as does biotype 1 (38, 48), but capsule, essential for development of infection in mice (7), is not required to infect eels when cells are intraperitoneally challenged (15). The pathology of the vibriosis is similar to that produced by V. anguillarum, which is hemolytic in nature (18). In the latter species, the ability to produce hemolytic anemia is related to its high iron requirement. Thus, pathogenic V. anguillarum strains have a welldeveloped iron-sequestering mechanism based on secretion of a siderophore which induces separation of plasma iron from transferrin. Iron complexes to the siderophore, and the complex is attached to a specific complex transporting outer membrane protein (OMP) for absorption into the bacterial cells (1).

MATERIALS AND METHODS Bacterial strains and growth conditions. A total of eight biotype 2 and four biotype 1 strains were used in this study (Table 1). The colonial morphology of all biotype 2 strains is opaque with the exception of strain NCIMB 2137, which is constitutively translucent and never reverts to the opaque morphology (15). European eel isolates were selected as representative of different epizootic outbreaks registered on an eel farm in Spain (10). All strains were routinely grown in tryptone soy broth or agar (Difco) at a final concentration of 1% NaCl (wt/vol) (TSB-1 or TSA-1) for 24 h at 258C. Stock cultures were kept in Marine broth (Difco) plus 20% (vol/vol) glycerol at 2808C. All glassware was soaked overnight in 6 M chloridic acid and rinsed extensively with distilled deionized water. For growth in iron-restricted conditions the minimal media M9 and M9 agar (M9A) (31) supplemented with 10 mM ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDHA; Sigma, St. Louis, Mo.) (M9-E and M9A-E, respectively) or with human transferrin (iron-free human apotransferrin; Sigma, Madrid, Spain) (M9-Tf and M9A-Tf) were used. Stock solutions of EDDHA and transferrin as well as iron-poor media were prepared as previously described (15). For some experiments iron-poor M9 medium (MM9) was produced by deferration with 8-hydroxyquinoline as described previously (3).

* Corresponding author. Mailing address: Departamento de Microbiologıá y Ecologıá, Facultad de Biologıá, Universidad de Valencia, Avda. Dr. Moliner 50, Burjassot, Valencia 46100, Spain. Phone: 6-3864389. Fax: 6-3894372. 928

V. VULNIFICUS BIOTYPE 2

VOL. 62, 1996

929

TABLE 1. Origin, colony type, and siderophore detection Strain(s)

Origin

Colony typea

Siderophore production CAS assayb

Arnow testc

Csa`ky testd

Biotype 2 E22, E39, E86, E105 E58, E116 ATCC 33149 NCIMB 2137

Diseased Diseased Diseased Diseased

European eel European eel Japanese eel Japanese eel

Opaque Opaque Opaque Translucent

1 1 1 1

1/2 1/2 1/2 1/2

11 1 1 1

Biotype 1 ATCC 27562 CDC7184 E109 TW1

Blood Human blood Gills of European eels Tank water of Spanish eel farm

Opaque Opaque Opaque Translucent

1 1 1 1

1/2 1 1/2 1/2

1 1 1 1

a

Original colony type. All the opaque strains are capsulated, and the translucent ones are acapsulated (15). Ratio of orange-halo diameter to colony diameter in CAS agar plates. 2, ,1.3; 1, 1.3 to 2.0. c For detection of catechols. 2, negative; 1/2, weakly positive (0.005 , A517.5 , 0.02); 1, positive (A517.5 . 0.02). d For detection of hydroxamates. 2, negative; 1, positive (0.01 , A520 , 0.06); 11, strongly positive (A520 . 0.06). b

Growth in serum. Eels were bled by venipuncture, and fresh serum was obtained by clotting blood from various animals at room temperature for 2 h and then at 48C overnight. Iron saturation levels of eel sera were evaluated with a commercial kit from Sigma which measures the unsaturated iron-binding capacity (15). Pooled serum was stored at 2208C until used. For some experiments fresh human serum, obtained as previously described (7), was also used. Isolated colonies on TSA-1 were inoculated overnight on M9A. Bacteria were harvested in sterile saline solution containing 1.5% NaCl (pH 7.5), washed, and adjusted up to 108 CFU/ml. Approximately 106 CFU was inoculated per ml of serum. Growth was monitored by measurement of optical density at 600 nm at different time intervals, and controls of viability were performed on TSA-1 after 9 and 24 h of incubation. Virulence assays with iron-pretreated eels. To evaluate the effect of iron pretreatment on the virulence of V. vulnificus biotype 2 for eels, animals were injected intraperitoneally with hemoglobin (bovine; Sigma, Madrid, Spain) (Hb), ferric ammonium citrate (FAC), or deferrioxamine B mesylate (Desferal; Sigma, Madrid, Spain) 24 h before bacterial infection (40). Stock solutions of Hb (1.25 mM), FAC (10 mM), and Desferal (0.25 g/liter) were prepared as previously described (7). The following amounts were administered per gram of body weight: (i) 9 mg of Fe as FAC, (ii) 0.8 mg of Fe as Hb, and (iii) 250 mg of Desferal. For each experiment, six elvers (average weight, 10 g) were injected with one of the bacterial doses, and two sets of animals, one inoculated with the appropriate iron solution and the other one inoculated with bacteria without iron pretreatment, were used as controls. Mortalities were recorded daily for 7 days, and virulence (LD50) for iron-overloaded animals was calculated according to the method of Reed and Mu ¨nch (29). Siderophore detection. Production of siderophores was investigated by using the universal assay of Schwyn and Neilands (33) as previously described (3). The ratio of (i) orange-halo diameter to colony diameter on chrome azurol S (CAS) agar plates and (ii) A630 of fresh and cell-free iron-poor media and sera after the addition of the CAS solution or CAS ‘‘shuttle’’ solution (33) were used for approximate quantification of the siderophore levels produced (3, 11). The colorimetric tests of Arnow (9) and Csa`ky (modified by Andrus et al. [8]) were used to detect phenolate- and hydroxamate-type siderophores in cell-free supernatants from bacterial cultures in iron-poor media and sera. Cross-feeding assays. The ability of V. vulnificus strains (test strains) to promote growth of other strains subjected to iron starvation (indicator strains) was tested in iron-poor artificial media and serum as previously described (3, 11). The iron chelators EDDHA and transferrin were added to media at the MICs established previously (15). Test cells were grown in M9-E or M9-Tf, and cell-free supernatants, positive for CAS assay, were pipetted onto 6-mm sterile filter paper disks (Oxoid) or added to sera in the proportion 1:10 (supplemented serum). After drying, disks were placed on M9A-E or M9A-Tf plates containing isolates of both biotypes as indicator strains. Control (serum plus iron-poor media in the proportion 10:1) and supplemented sera were immediately inoculated with the indicator strain, and growth was monitored by measurement of optical density as described above. Bioassays were also performed with mutants deficient in the production of known siderophores. The mutant V. anguillarum 775::Tn1-5, anguibactin deficient, and the wild strain 775 were used for anguibactin-like siderophore detection (1). Salmonella typhimurium enb-1 and enb-7 mutants, deficient for enterobactin biosynthesis, and the positive control S. typhimurium LT2 were used for enterobactin bioassays (28). Escherichia coli LG1522, deficient for aerobactin biosynthesis, and the positive control E. coli LG1315 were used for aerobactin detection (43). All experiments were conducted with M9A-E plates under the conditions established previously (3). Arthrobacter flavescens JG-9, now desig-

nated Aureobacterium flavescens JG-9, a hydroxamate auxotroph, was used to evaluate the production of hydroxamate-type siderophores other than aerobactin (47). These bioassays were performed by the method of Yancey and Finkelstein (47) using Desferal (sterile disks impregnated with 20 ml of a solution containing 0.25 mg of Desferal per ml of distilled water) as the positive control and TSA-1 as the culture medium. Absorbance spectra and paper chromatography. Cell-free supernatants of cultures grown in iron-restricted media were chromatographed through Whatman no. 1 filter paper (3MM) as previously described (3, 11) by using butanolacetic acid-water as solvent (60:15:25 [vol/vol/vol]). Chromatograms were made in duplicate. Papers were air dried, and spots of iron-binding compounds were visualized by illumination with UV light and sprayed with CAS solution (33) or 1% FeCl3 z 6H2O in 0.01 M HCl to determine the presence of iron-binding compounds. Cell-free supernatants were scanned between 200 and 500 nm, and absorbance spectra were determined with a DU-7 spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). Analysis of OMPs and LPSs. Lipopolysaccharides (LPSs) and OMPs were analyzed in order to study the effect of iron restriction on cell envelope composition. Strains were grown in M9 plus 10 mM FeCl3 (M9-Fe), M9-E, and M9-Tf for 24 h, and then LPSs and OMPs were extracted as described by Amaro et al. (6) and Biosca et al. (14), respectively. LPS and OMP samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (21) using a separation gel of 12.5% acrylamide. Twenty microliters of the LPS sample and approximately 60 mg of protein were applied per slot. LPS bands were visualized by immunoblotting with antiserum against strain E22 by the procedure described by Amaro et al. (6), while the protein bands were stained with Coomassie brilliant blue. Molecular masses of the protein bands were determined as described before (3). Dot blot assay for detection of the transferrin receptor. In order to determine whether V. vulnificus possesses membrane receptors for human transferrin, dot blot assays were performed as described previously (32) by using peroxidaseconjugated human transferrin (Jackson Immunoresearch Laboratories). Briefly, 2-ml samples of bacterial cells grown to late log phase in M9-E and M9-Fe were directly applied to cellulose acetate paper (0.45-mm-pore-size Ha paper; Millipore) and allowed to dry. Nonspecific protein binding sites on the paper were blocked for 1 h with Tris-buffered saline (TBS) (50 mM Tris [pH 7.5], 150 mM NaCl) containing 0.5% skim milk, 0.001% Tween 20, and 0.01 thimerosal (TTBS). The paper was washed with TBS prior to addition of TTBS containing 500 ng of conjugated transferrin per ml. The binding mixture was incubated at 378C for 1 h, the solution was removed, and the paper was washed three times with TTBS before being developed with a chloronaphthol-hydrogen peroxide substrate mixture (HRP reagent; Bio-Rad) at room temperature for 5 to 10 min. Neisseria meningitidis B16B6 was used as a positive control (32). Bioassay for iron uptake from transferrin. In order to determine whether growth from human transferrin requires cell-protein contact, an assay using dialysis tubing containing bacterial cells was carried out. Dialysis bags (Servapor 1 with a 6,000- to 8,000-Da cutoff) were prepared by the procedure outlined by Sambrook et al. (31). Bacterial cultures in M9 were harvested, and pellets were washed in MM9 salts up to a concentration of 106 CFU/ml. A volume of 2 ml of a bacterial suspension was sequestered inside dialysis bags which were completely immersed in flasks containing M9-Tf. Dialysis tubing, running along the bottom of the flask, was long enough to ensure that both ends could be closed with two flat knots at the mouth of the flask (2). This prevented bacteria from leaking into the external medium. In these conditions, bacteria will grow if they

930

BIOSCA ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 2. Effects of pretreatment with Desferal, ferric ammonium citrate (iron), and Hb on the susceptibilities of eels to infection with different V. vulnificus strains LD50 (CFU/fish) Pretreatmenta (mgb)

None Desferal (250) Iron (9) Hb (0.8)

Biotype 1

Biotype 2, E58

ATCC 27562

CDC 7184

E109

9.4 3 104 ,2.1 3 102 ,2.1 3 102 ,2.1 3 102

.1.7 3 108 .1.2 3 108 .1.2 3 108 .1.2 3 108

.2.0 3 108 .3.8 3 108 .3.8 3 108 .3.8 3 108

.5.2 3 108 .2.8 3 107 .2.8 3 107 .2.8 3 107

a Intraperitoneal inoculation of eels with an iron source 24 h before intraperitoneal inoculation with bacteria. b Of iron per gram of body weight.

produce iron chelators able to cross through the dialysis bag and sequester iron from transferrin (2). Experiments were done in triplicate. Control flasks containing sterile M9 medium without transferrin were included to ensure that the dialysis tubing itself did not interfere with growth. Negative controls were made by inoculating the strain N. meningitidis B16B6, which grows from iron-transferrin only by protein-cell contact (27, 35). Bacterial counts, the presence of siderophores both inside and outside the dialysis bags, and changes in the outer membrane protein profiles were evaluated after 24 h of incubation as described before.

RESULTS Iron availability and virulence. After a preliminary screening of twenty European strains (10), we found the majority to be highly virulent for eels (LD50, 101 to 103 CFU per fish) with the exception of some isolates from the same epizootic outbreak which were moderately virulent (around 105 CFU per fish) (data not shown). We chose strain E58 as representative of this group of moderately virulent strains and assessed the role of Hb, FAC, and Desferal as adjuvants for the pathogenicity of biotype 2. No death was observed in controls that received Hb, FAC, or Desferal alone. These iron sources and the siderophore Desferal reduced the mean lethal dose of strain E58 from 105 to less than 102 CFU per fish in all experiments (Table 2) and also the time it took them to die: all eels died less than 24 to 48 h postinjection. In all experiments, the inoculated strain was recovered as a pure culture from internal organs. Biotype 1 strains, used for comparative purposes, remained avirulent even for iron-pretreated animals (Table 2). Siderophore detection. We have previously demonstrated the ability of V. vulnificus biotype 2 to grow under the restricted iron conditions imposed by EDDHA or transferrin (15). In this study, the method of Schwyn and Neilands was used to relate growth in both iron-deficient medium and serum to siderophore production. After testing V. anguillarum 775:: Tn1-5, deficient for anguibactin production (1), we considered a ratio between orange-halo and colony diameters on CAS agar plates of less than 1.3 as a negative result. All biotype 2 strains gave positive results, irrespective of their virulence degree and possession of capsule, and showed a zone ratio of around 1.8 (Table 1). Similar results were obtained with strains belonging to biotype 1. Siderophore detection assays using the cell-free supernatant fluids of MM9 medium and the CAS solution for liquid media confirmed the results obtained with CAS agar plates. Siderophore presence was also investigated in cell-free sera from humans and eels inoculated with V. vulnificus biotype 2. All biotype 2 strains were strongly positive in human serum and slightly positive in eel serum (data not shown), results which correlated with the saturation levels of the transferrins: approximately 75 and 25% for eel and human sera, respectively. After the addition of CAS solution, the color

changed from blue to orange within hours, this change being accelerated when the CAS shuttle solution was used (less than 20 min). These results suggested that the siderophores excreted were mainly hydroxamic (33). Then, these samples were further examined for the detection of catechols and hydroxamates by using the Arnow and Csa`ky tests, respectively. Since the reagents precipitated serum components, we used as samples the cell-free supernatants of M9-E and M9-Tf, which were positive for the CAS assay. All biotype 2 strains, as well as controls of biotype 1, gave positive results in the Csa`ky test, suggesting the presence of hydroxamate-type siderophores (Table 1). A weak positive result was achieved in the Arnow test, with the sole exception of the clinical isolate of biotype 1 CDC7184, which gave a strong positive response (Table 1). These data coincide with previous results obtained by other researchers working with this strain (39). No apparent differences in siderophore production were observed among translucent and opaque strains or among biotype 2 strains with regard to their virulence degree (Table 1). Cross-feeding assays. The secretion and utilization of biologically active iron uptake compounds were also assayed. All cell-free supernatants of M9-E and M9-Tf of V. vulnificus biotype 2 isolates were able to stimulate growth of homologous and heterologous iron-starved strains irrespective of their origins (water, clinical, or eel samples), geographic distributions, or biotypes (Table 3). Cell-free supernatants in iron-poor media also stimulated bacterial growth in both human and eel sera, the effect obtained in human serum being more apparent (Fig. 1). As we have remarked before, the iron-chelating effect of the latter serum is stronger because of the lower degree of saturation of transferrins. In order to determine the biological activity of siderophores, cross-feeding assays were performed with different mutants of V. anguillarum, S. typhimurium, E. coli, and A. flavescens deficient in the anguibactin-, enterobactin-, aerobactin-, and hydroxamic acid-mediated systems, respectively. All biotype 2 strains were negative for anguibactin, enterobactin, and aerobactin production but were able to stimulate growth of the auxotrophic mutant of A. flavescens, indicating production of a hydroxamate-type siderophore different from aerobactin. All strains were also able to use Desferal to reverse the iron limitation imposed by the synthetic iron chelator EDDHA (Table 3). Although our strains did not produce enterobactin, they were able to stimulate the growth of the mutant S. typhimurium enb-7 that can utilize 2,3-dihydroxybenzoic acid (2,3-DHBA), a precursor for enterobactin biosynthesis as well as some hydroxamate-type siderophores (30). Similar results were obtained by using biotype 1 strains for comparative purposes (Table 3). Utilization of human transferrin as the only iron source. To determine whether cell-protein contact, soluble iron-chelating compounds, or both are necessary for growth from iron-transferrin, we carried out two kinds of experiments: dot blot assays for transferrin receptor activity and the dialysis tubing assay. In the first, none of the strains tested, irrespective of their biotype, origin, and presence of capsule, presented specific receptors for human transferrin even when cells were grown in iron-restricted conditions (Fig. 2). By using the second assay with selected strains of both biotypes (E22, E39, E86, E105, ATCC 27562, E109, and TW1), we confirmed that all strains were able to grow without direct contact with the glycoprotein. After 24 h of incubation, bacterial counts increased from (8.0 6 1.5) 3 105 to (9.7 6 1.3) 3 107 and from (9.2 6 0.5) 3 105 to (3.5 6 0.4) 3 107 in the case of biotype 2 and biotype 1 cells, respectively. We confirmed the presence of siderophores by the Arnow and Csa`ky tests inside and outside dialysis bags. These siderophores were only of the hydroxamate type in the


VOL. 62, 1996

931

TABLE 3. Cross-feeding assays of V. vulnificus strains with various indicator strains Growth witha: Test strain(s) or substance

V. anguillarumb 775 R1 S2

S. typhimuriumc enb-1

enb-7

E. colid LG1522

A. flavescense JG-9

V. vulnificus Biotype 2f

Biotype 1g

Biotype 2: ATCC 33149, E22, E39, E58, E86, E105, E116, NCIMB 2137

2

2

1/2

2

1

1

1

Biotype 1: ATCC 27562, CDC7184 E109, TW1

2

2

1/2

2

11

11

11

Controls V. anguillarum 775 S. typhimurium LT2 E. coli LG1315 Desferal

11 2 2 NT

2 11 2 NT

1 1 1 NT

2 2 1 NT

2 2 2 1

NTh NT NT 1

NT NT NT 1

a Growth of indicator strain in M9A-E or M9A-Tf around filter paper disks soaked with supernatant fluids from cultures of test strains in M9-E or M9-Tf. Diameter of the zone of growth: 11, .15 mm; 1, ,15 mm. 1/2, result variable depending on the strain; 2, no growth. b Bioassay for the detection of anguibactin. c Bioassay for the detection of enterobactin. d Bioassay for the detection of aerobactin. e Bioassay for the detection of hydroxamate compounds other than aerobactin. f Strains E22, E39, E58, E86, E105, and E116. g Strains ATCC 27562, CDC7184, E109, and TW1. h NT, not tested.

case of biotype 2 strains (A520 of 0.06 to 0.2 depending on the strain and experiment) and of both types in the case of the biotype 1 strains used for comparative purposes. No growth was observed in flasks inoculated with N. meningitidis B16B6, which needs direct contact with transferrin to grow. Paper chromatography and absorbance spectra. Since chemical and biological tests from samples of dialysis tubing indicated the presence of hydroxamic compounds, samples of strains E39 and E86 were analyzed by paper chromatography in appropriate solvent systems (3, 11). Three spots with Rfs of about 0.13, 0.35, and 0.6 were blue fluorescent under UV light, but only the spot with the Rf of 0.13 was clearly iron reactive after being sprayed with CAS or FeCl3 solution. The absorption spectra of cell-free supernatants showed that the hydroxamate compounds absorbed mainly in the UV range, with a narrow peak at 262 nm and a smaller and broader peak at 330 nm. Membrane alterations in response to iron availability. We studied the alterations in the membrane of V. vulnificus biotype 2 in response to iron starvation. Isolates of biotype 1 were

FIG. 1. Growth of the V. vulnificus biotype 2 strain E58 in eel and human sera with or without cell-free supernatant fluids (10% [vol/vol]) from strain E39 grown in an iron restriction medium (M9-E). ■, eel serum; h, eel serum plus cell-free supernatant; }, human serum; {, human serum plus cell-free supernatant.

included for comparative purposes. The OMP profiles obtained in iron-rich and iron-deficient media confirmed that all strains synthesize iron-regulated OMPs (IROMPs) of high molecular masses (Fig. 3 and 4). In membrane profiles from biotype 2 isolates, two new proteins of identical molecular masses (84 and 72 kDa) were detected when cells were grown in iron-deficient conditions (Fig. 3). Apparently, the same IROMPs were observed independently of the kind of iron chelator used (the biological iron chelator, human transferrin, or the synthetic one, EDDHA) (Fig. 5) and of the kind of experiment performed; the same bands were seen when cells were grown inside dialysis bags (data not shown). Variable results were obtained with biotype 1 strains. Each strain presented a specific pattern of IROMPs, whose molecular masses ranged from 86 to 69 kDa (Fig. 4). The majority of strains synthesized two new OMPs in response to iron starvation, except strain TW1, which presented only one new protein of around 69 kDa. These proteins were observed in the electrophoretic profiles obtained from biotype 1 cells grown in the presence of either of the iron chelators used (Fig. 5). Changes in the LPS profile in response to iron starvation have been reported for Bordetella pertussis (2). Regarding V. vulnificus biotype 2, no differences in the LPS profiles of cells grown in iron-rich (M9-Fe) medium and those grown in iron-

FIG. 2. Dot blot assay for detection of the transferrin receptor. Two-microliter aliquots of bacterial cells grown to late log phase in M9-E and M9-Fe were directly applied to cellulose acetate paper. N. meningitidis B16B6 was used as positive control. A2 and A4, strain E39 in M9-Fe; B1 and B3, E39 in M9-E; D3 and D5, strain E86 in M9-Fe; F2 and F4, E86 in M9-E; H4, strain CDC7184 in M9-E; I1, CDC7184 in M9-Fe; I5, N. meningitidis B16B6 in M9-Fe.

932

BIOSCA ET AL.

FIG. 3. SDS-PAGE of OMPs of V. vulnificus biotype 2. Lane A1, strain E39 in M9-Fe; lane A2, E39 in M9-E; lane B1, strain E86 in M9-Fe; lane B2, E86 in M9-E. The molecular masses of IROMPs are indicated.

starved (M9-E and M9-Tf) media were observed (Fig. 6). Similar results were obtained for biotype 1 strains used for comparative purposes (Fig. 6). DISCUSSION Iron availability and degree of virulence. V. vulnificus biotype 1 is an opportunistic human pathogen whose virulence degree correlates directly with the availability of iron in body fluids (44). Correlation between virulence degree and iron availability is easily demonstrated in the laboratory by a reduction of 50% in lethal dose for iron-overloaded mice (44). Biotype 2 comprises the pathogenic strains for eel. This biotype was considered an obligate eel pathogen until we demonstrated that it is able to survive in water and use it as a transmission route (5). The fact that it can be an opportunistic pathogen for humans has been highlighted after the isolation of an indole-negative variant (this characteristic is positive for biotype 1 and negative for biotype 2 [4, 10, 12, 13, 42]) from a patient with a septicemic infection (19). This is consistent with the virulence for mice displayed by strains belonging to this biotype (7, 42), the degree of which is dependent on the avail-

FIG. 4. SDS-PAGE of OMPs of V. vulnificus biotype 1. Lane A1, strain ATCC 27562 in M9-Fe; lane A2, ATCC 27562 in M9-E; lane B1, strain TW1 in M9-Fe; lane B2, TW1 in M9-E. The molecular masses of IROMPs are indicated.


FIG. 5. SDS-PAGE of OMPs of V. vulnificus. Lane A1, biotype 2 strain E39 in M9-E; lane A2, E39 in M9-Tf; lane B1, biotype 1 strain ATCC 27562 in M9-E; lane B2, ATCC 27562 in M9-Tf. The molecular masses of IROMPs are indicated.

ability of free iron in body fluids (7). In fact, biotype 2 can use hemin, Hb, and Desferal (combined with iron from the iron chelators that contain minimal media) as iron sources in vitro and in vivo (mice) (7). The majority of the Spanish strains of this biotype are highly efficient eel pathogens with the exception of some moderately virulent strains, a representative of which has been used in this study. After evaluating the effects of these iron sources on the virulence degree of this strain, we found that they greatly increased the susceptibility of eels to vibriosis, probably because of the rapid growth of cells in body fluids. These data demonstrate that the relationship between iron availability and virulence degree of V. vulnificus biotype 2 is manifested in both fish and mammals (mice). Biotype 1 strains remained avirulent even for eels pretreated with iron, confirming that this biotype is not pathogenic for eels. Siderophores and iron acquisition mechanisms. Highly virulent strains of V. vulnificus biotype 2 are able to grow rapidly in fish without any additional iron source (4, 15). This study suggests that siderophore-mediated iron uptake may be, in part, responsible for this ability to grow rapidly in fish. Con-

FIG. 6. SDS-PAGE of LPSs of V. vulnificus. Lane A1, biotype 2 strain E39 in M9-Fe; lane A2, E39 in M9-E; lane A3, E39 in M9-Tf; lane B1, biotype 1 strain ATCC 27562 in M9-Fe; lane B2, ATCC 27562 in M9-E; lane B3, ATCC 27562 in M9-Tf. Gels were stained by immunoblotting with the homologous antiserum.

VOL. 62, 1996

cerning this property, there is a similarity to another systemic pathogen affecting fish, V. anguillarum, in which a high-affinity iron-sequestering system has been demonstrated as an important virulence attribute (1). However, unlike V. anguillarum, V. vulnificus biotype 2 is able to infect other species of fish (10), mice (7), and probably humans (19), so the iron acquisition mechanism displayed must be equally efficient in all these hosts. In a previous paper (15) we showed that V. vulnificus biotype 2 was able to grow in the presence of the iron chelator EDDHA or transferrin as well as in eel serum. The siderophore detection assays used in the present study demonstrated that this biotype is able to produce both hydroxamate- and, apparently, catechol-type siderophores. Siderophores with a hydroxamic nature are the main ones to be detected in iron-poor media and sera. The supernatant fluids of cultures of hydroxamate-producing strains showed an iron-reactive spot in paper chromatograms and absorbance spectra similar to that described for hydroxamates belonging to the aerobactin-like class, which are produced by other vibrios (3, 11). Biological activity of the siderophores, tested by bioassays with mutants deficient in known siderophores, showed that V. vulnificus biotype 2 does not produce either enterobactin or anguibactin, or aerobactin, but produces hydroxamates of an unknown nature, able to stimulate A. flavescens JG-9. The production of phenolates (although the amount is small) cannot be dismissed because of the positive response, although weak, both in the Arnow test and in bioassays with S. typhimurium enb-7. Apart from enterobactin, the latter mutant is able to use its biosynthetic precursor 2,3-DHBA as well as some hydroxamates (30). The simultaneous production of both phenolates and hydroxamates of an unknown nature has previously been reported for V. vulnificus biotype 1 (8, 36, 46). The phenolate has recently been characterized, and the name ‘‘vulnibactin’’ has been proposed (24). In fact, both kinds of siderophores were produced by the strains of biotype 1 used for comparative purposes which gave the same results in the bioassays. Isolates of both biotypes were able to stimulate each other, suggesting that these substances are relatively homogeneous within the species. From the results obtained with iron-overloaded animals it seems that the differences in virulence degree are related to the efficiency of the iron-sequestering mechanism. V. vulnificus biotype 2 is able to use mechanisms for iron acquisition other than siderophore production, such as secretion of hemolysin and use of hemin and Hb as iron sources (7). The importance of such mechanisms in the progress of the infectious process has yet to be investigated. In vertebrates, the small amount of extracellular iron in body fluids is attached to the host’s iron-binding and transport proteins, i.e., transferrin in serum and lymph, thereby effectively creating an iron-restricted environment for extracellular microorganisms (27). During infection, eels further reduce the total amount of extracellular iron, producing hypoferremia (20). Extracellular bacterial pathogens can overcome this bacteriostatic effect by means of siderophores, which compete with the host iron-binding proteins, or through the possession of OMP receptors that recognize the complex transferrin-iron (27). Through previous studies we know that biotype 2 cells efficiently grow in eel serum and that they can be isolated from blood samples during the infection (10, 15). In this work we detected siderophores, mainly hydroxamates, in both cell-free M9-Tf and serum. To clarify whether V. vulnificus biotype 2 can use these siderophores for iron uptake from transferrin or if it needs contact with iron-transferrin to grow, we used the membrane dialysis assay and the dot blot assay for transferrin receptor detection. Since transferrins, even from phylogeneti-


933

cally distant species, have considerable sequence homology (16) and this biotype is also an opportunistic pathogen for humans (19), we used purified human transferrin in these experiments. All strains lacked a specific receptor for human transferrin and were able to grow from iron-transferrin by producing hydroxamates which seemed to compete efficiently for iron with the glycoprotein. Similar results were obtained with biotype 1 strains, although in this case phenolates were also detected depending on the strain. Andrus et al. (8) found that the hydroxamates produced by V. vulnificus biotype 1 were atypical but they had a greater iron-chelating activity than the phenolates produced by this organism. High levels of hydroxamate-type siderophores were also produced and secreted by V. vulnificus biotype 2 strains in all assays carried out during the study (growth in serum, cross-feeding assays, and transferrin assay). These results are consistent with all known examples of bacteria that produce two kinds of siderophores simultaneously in culture media; they excrete hydroxamates during infection (17). The hydroxamates produced by V. vulnificus biotype 2 can efficiently compete for iron with transferrins, and therefore they could be used as an iron acquisition mechanism during bacterial infection. This result is consistent with the opportunistic-pathogen nature of this biotype, which is able to infect different hosts: eels (12), sea bass (10), turbot (10), mice (7), and humans (19). Cell surface and iron acquisition. The study of alterations in external envelopes of V. vulnificus biotype 2 showed that two new proteins of high Mrs were synthesized in response to iron starvation. The same proteins were induced irrespective of the chelator used to achieve iron restriction conditions. Although their precise function is unknown, they are probably involved in iron transport from ferric siderophores, as in other vibrios (1). Apparently, IROMPs of identical Mrs were expressed by all biotype 2 strains irrespective of their geographic origins, in agreement with the similarity previously found among their OMP and LPS profiles (6, 14). In fact, biotype 2 isolates belong to the same serogroup and present identical LPS patterns (6). In contrast, different IROMPs were synthesized by biotype 1 strains used for comparative purposes, in accordance with the variability in OMP and LPS profiles previously found for this biotype (6, 14, 46). At least seven different serogroups have been described for biotype 1 strains (34). These results together with the greater variability in siderophore production suggest that more than one siderophore-dependent iron uptake mechanism may be expressed by biotype 1 strains. Changes in LPS in response to iron starvation have been reported for B. pertussis and have been related to the pathogenesis of this organism (2). Similar studies had not been carried out in the species V. vulnificus. No alterations were observed in the LPS profiles of V. vulnificus biotype 2 strains under iron restriction conditions, and similar results were obtained with biotype 1 strains used for comparative purposes. In conclusion, changes in external iron concentration seem to affect external membranes in their protein composition. It has been suggested that capsule is essential for iron acquisition from transferrin by biotype 1 cells; only opaque cells are able to grow in the presence of human transferrin and resist the bactericidal and bacteriostatic effect of human serum (38). This observation is questioned by Wright et al. (45), who demonstrated that an acapsular mutant was able to grow from human transferrin. In a previous study, we demonstrated that capsule confers to biotype 2 cells protection against complement of human serum but it is not essential for growth in iron-poor media, fresh eel serum, or heat-inactivated human serum (7, 15). Since the translucent strain NCIMB 2137 is able to grow in iron-restricted conditions by means of the expres-

934

BIOSCA ET AL.


sion of the same siderophore-dependent iron-acquisition mechanism, results in this work confirm our previous ones and are in accordance with those obtained by Wright et al. for biotype 1 (45). In conclusion, V. vulnificus biotype 2 expresses an iron uptake mechanism which is dependent on siderophores, and the chelator responsible for the iron acquisition from transferrins is most probably the hydroxamic type. The importance of siderophores in the pathogenicity of V. vulnificus biotype 2 is not quite clear, given that this pathogen is also hemolytic (15) and able to use hemin and hemoglobin as iron sources in vivo (7). In any case, for an invasive organism the ability to synthesize siderophores is undoubtedly an advantage to its survival and growth in body fluids. From previous results we know that this pathogen is able to infect a wide range of animal hosts other than eels (10). As the pathogen is opportunistic, the siderophore-dependent iron uptake mechanism may play an important role in iron acquisition under aerobic conditions in the environment and, therefore, contribute to the survival of the pathogen outside its host.

ACKNOWLEDGMENTS This work was partially supported by grants AGF95-1085-CO2-O1 from Comisio ń Interministerial de Ciencia y Tecnologıá of Spain and C412 from IMPIVA, Valencia, Spain. E. G. Biosca thanks Consellerıá de Cultura, Educacio ń y Ciencia de la Generalitat Valenciana, and B. Fouz thanks Consellerıá de Educacio ń, Xunta de Galicia, for their respective predoctoral fellowships. REFERENCES 1. Actis, L. A., S. A. Potter, and J. W. Crosa. 1985. Iron-regulated outer membrane protein OM2 of Vibrio anguillarum is encoded by virulence plasmid JM1. J. Bacteriol. 161:736–742. 2. Agiato, L. A., and D. W. Dyer. 1992. Siderophore production and membrane alterations by Bordetella pertussis in response to iron starvation. Infect. Immun. 60:117–123. 3. Amaro, C., R. Aznar, E. Alcaide, and M. L. Lemos. 1990. Iron-binding compounds and related outer membrane proteins in Vibrio cholerae non-O1 strains from aquatic environments. Appl. Environ. Microbiol. 56:2410–2416. 4. Amaro, C., E. G. Biosca, C. Esteve, B. Fouz, and A. E. Toranzo. 1992. Comparative study of phenotypic and virulence properties in Vibrio vulnificus biotypes 1 and 2 obtained from a European eel farm experiencing mortalities. Dis. Aquat. Org. 13:29–35. 5. Amaro, C., E. G. Biosca, B. Fouz, E. Alcaide, and C. Esteve. 1995. Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels. Appl. Environ. Microbiol. 61:1133–1137. 6. Amaro, C., E. G. Biosca, B. Fouz, and E. Garay. 1992. Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains of Vibrio vulnificus biotypes 1 and 2 using silver staining and immunoblotting. Curr. Microbiol. 25:99–104. 7. Amaro, C., E. G. Biosca, B. Fouz, A. E. Toranzo, and E. Garay. 1994. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect. Immun. 62:759–763. 8. Andrus, C. R., M. A. Walter, J. H. Crosa, and S. M. Payne. 1983. Synthesis of siderophores by pathogenic Vibrio species. Curr. Microbiol. 9:209–214. 9. Arnow, L. E. 1937. Colorimetric determination of the components of 3,4dihydroxyphenylalanine-tyrosine mixtures. J. Biol. Chem. 118:531–541. 10. Biosca, E. G. 1994. Ph.D. thesis. Universidad de Valencia, Valencia, Spain. 11. Biosca, E. G., and C. Amaro. 1991. Siderophore and related outer membrane proteins in Vibrio spp. which are potential pathogens of fish and shellfish. J. Fish Dis. 14:249–263. 12. Biosca, E. G., C. Amaro, C. Esteve, E. Alcaide, and E. Garay. 1991. First record of Vibrio vulnificus biotype 2 from diseased European eels (Anguilla anguilla). J. Fish Dis. 14:103–109. 13. Biosca, E. G., C. Esteve, E. Garay, and C. Amaro. 1993. Evaluation of the API 20E system for the routine diagnosis of the vibriosis produced by Vibrio vulnificus biotype 2. J. Fish Dis. 16:79–82. 14. Biosca, E. G., E. Garay, A. E. Toranzo, and C. Amaro. 1993. Comparison of the outer membrane protein profile of Vibrio vulnificus biotypes 1 and 2. FEMS Microbiol. Lett. 107:217–222. 15. Biosca, E. G., H. Llorens, E. Garay, and C. Amaro. 1993. Presence of a capsule in Vibrio vulnificus biotype 2 and its relationship to virulence for eels.

Infect. Immun. 61:1611–1618. 16. Brock, J. H. 1985. Transferrins, p. 183–262. In P. M. Harrison (ed.), Metalloproteins, part II. Macmillan, New York. 17. Cox, C. D. 1989. Importance of iron in bacterial virulence, p. 207–246. In T. J. Beveridge and R. J. Doyle (ed.), Metal ions and bacteria. John Wiley and Sons, Inc., New York. 18. Hjeltnes, B., and R. J. Roberts. 1993. Vibriosis, p. 109–122. In V. Ingris, R. J. Roberts, and N. R. Bromage (ed.), Bacterial diseases of fish. Blackwell Scientific Publications, London. 19. Jan-Veenstra, P., J. P. G. M. Rietra, C. P. Stoutenbeek, J. M. Coster, H. H. W. De Hier, and S. Dirsks-go. 1992. Infection by an indole-negative variant of Vibrio vulnificus transmitted by eel. J. Infect. Dis. 16:209–210. 20. Kakuta, I., and S. Murachi. 1993. Changes in iron content in eels (Anguilla japonica) infected with atypical Aeromonas salmonicida. Comp. Biochem. Physiol. 105:275–278. 21. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 277:680–685. 22. Morris, J. G., A. C. Wright, L. M. Simpson, P. K. Wood, D. E. Johnson, and J. D. Oliver. 1987. Virulence of Vibrio vulnificus: association with utilization of transferrin-bound iron, and lack of correlation with levels of cytotoxin or protease production. FEMS Microbiol. Lett. 40:55–59. 23. Muroga, K., Y. Jo, and M. Nishibuchi. 1976. Pathogenic Vibrio isolated from cultured eels. I. Characteristics and taxonomic status. Fish Pathol. 11:141– 145. 24. Okujo, N., M. Saito, S. Yamamoto, T. Yoshida, S. Miyoshi, and S. Shinoda. 1994. Structure of vulnibactin, a new polyamine-containing siderophore from Vibrio vulnificus. Biometals 7(2):109–116. 25. Oliver, J. D., R. A. Warner, and D. R. Cleland. 1982. Distribution and ecology of Vibrio vulnificus and other lactose-fermenting marine vibrios in coastal waters of the southeastern United States. Appl. Environ. Microbiol. 44:1404–1414. 26. Oliver, J. D., R. A. Warner, and D. R. Cleland. 1983. Distribution of Vibrio vulnificus and other lactose-fermenting vibrios in the marine environment. Appl. Environ. Microbiol. 45:985–998. 27. Otto, B. R., A. M. J. J. Verweij-van Vught, and D. M. MacLaren. 1992. Transferrins and heme-compounds as iron sources for pathogenic bacteria. Crit. Rev. Microbiol. 18:217–233. 28. Pollack, J. R. B., B. N. Ames, and J. B. Neilands. 1970. Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin. J. Bacteriol. 104:635–639. 29. Reed, M. J., and M. Mu ¨nch. 1938. A simple method for estimating fifty percent endpoints. Am. J. Hyg. 27:493–497. 30. Reissbrodt, R., and W. Rabsch. 1988. Further differentiation of Enterobacteriaceae by means of siderophore pattern analysis. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 268:306–317. 31. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 32. Schryvers, A. B., and L. J. Morris. 1988. Identification and characterization of the transferrin receptor from Neisseria meningitidis. Mol. Microbiol. 2:281–288. 33. Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem. 160:47–56. 34. Shimada, T., and R. Sakazaki. 1984. On the serology of Vibrio vulnificus. Jpn. J. Med. Sci. Biol. 37:241–246. 35. Simonson, C., D. Brener, and I. W. DeVoe. 1982. Expression of a high-affinity mechanism for acquisition of transferrin iron by Neisseria meningitidis. Infect. Immun. 36:107–113. 36. Simpson, L. M., and J. D. Oliver. 1983. Siderophore production by Vibrio vulnificus. Infect. Immun. 41:644–649. 37. Simpson, L. M., and J. D. Oliver. 1987. Ability of Vibrio vulnificus to obtain iron from transferrin and other iron-binding proteins. Curr. Microbiol. 15: 155–157. 38. Simpson, L. M., V. K. White, S. F. Zane, and J. D. Oliver. 1987. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect. Immun. 55:269–272. 39. Stelma, G. N., A. L. Reyes, J. T. Peeler, C. H. Johnson, and P. L. Spaulding. 1992. Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus. Appl. Environ. Microbiol. 58:2776–2782. 40. Takaji, I., and H. Wakabayashi. 1990. Relationship between iron acquisition ability and virulence of Edwardsiella tarda, the etiological agent of paracolo disease in Japanese eel Anguilla japonica. The 2nd Asian Fisheries Forum. Asian Fisheries Society, Manila, Philippines. 41. Tamplin, M., G. E. Rodrick, N. J. Blake, and T. Cuba. 1982. Isolation and characterization of Vibrio vulnificus from two Florida estuaries. Appl. Environ. Microbiol. 44:1466–1470. 42. Tison, D. L., M. Nishibuchi, J. D. Greenwood, and R. J. Seidler. 1982. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl. Environ. Microbiol. 44:640–646. 43. Williams, P. H. 1979. Novel iron uptake system specified by Col V plasmids: an important component in the virulence of invasive strains of Escherichia coli. Infect. Immun. 26:925–932.

VOL. 62, 1996 44. Wright, A., L. M. Simpson, and J. D. Oliver. 1981. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect. Immun. 34:503–510. 45. Wright, A., L. M. Simpson, J. D. Oliver, and J. G. Morris. 1990. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect. Immun. 58:1769–1773. 46. Wright, A., L. M. Simpson, K. Richardson, D. R. Maneval, Jr., J. D. Oliver, and J. G. Morris. 1986. Siderophore production and outer membrane pro-


935

teins of selected Vibrio vulnificus strains under conditions of iron limitation. FEMS Microbiol. Lett. 35:255–260. 47. Yancey, R. J., and R. A. Finkelstein. 1981. Siderophore production by pathogenic Neisseria spp. Infect. Immun. 32:600–608. 48. Yoshida, S. I., M. Ogawa, and Y. Mizuguchi. 1985. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect. Immun. 47:446–451.